in situ Immuno-PCR: A Newly Developed Method for Highly Sensitive Antigen Detection in situ

Immunocytochemistry and in situ hybridization—the in situ staining approaches for detection of antigens, DNA, or RNA in intact cells and tissue sections— are common and powerful tools for biological and biomedical research and clinical diagnostics. By combining the exponential amplification power of the polymerase chain reaction (PCR) with in situ hybridization, in situ PCR and in situ reverse-transcription-polymerase chain reaction (RT-PCR) allows the detection of low copy numbers of nucleic acids in situ (1 –3 ). However, with routine immunocytochemistry, detection of the minute numbers of target molecules accessible with in situ PCR or in situ RT-PCR is difficult. Immuno-PCR, which uses PCR amplification to increase the signal of immunoassays, exhibits high sensitivity and permits the detection of proteins at the level of a few hundred molecules (4 –5 ). By combining the high sensitivity of in situ PCR amplification with the versatility and high specificity of immunocytochemistry, we have developed a new method known as in situ immuno-PCR to detect antigens at low levels in intact cells or tissue sections (6 ). The concept of in situ immuno-PCR is illustrated in Fig. 1 . Our study demonstrated that in situ immuno-PCR is more sensitive for the detection of Hepatitis B-specific antigen (HBsAg) in situ compared with other signal-amplification systems (Fig. 2 ). in situ immuno-PCR may be the only technique available to detect minute quantities of biological macromolecules such as proteins, carbohydrates, and lipids in intact cells or tissue sections. This new method should find wide application in biological research and clinical diagnostics.